Mycobacterium tuberculosis impairs human memory CD4+ T cell recognition of M2 but not M1-like macrophages.

Direct recognition of Mycobacterium tuberculosis (Mtb)-infected cells is required for protection by CD4+ T cells. While impaired T cell recognition of Mtb-infected macrophages was demonstrated in mice, data are lacking for humans. Using T cells and monocyte-derived macrophages (MDMs) from individuals with latent Mtb infection (LTBI), we quantified the frequency of memory CD4+ T cell activation in response to autologous MDMs infected with virulent Mtb. We observed robust T cell activation in response to Mtb infection of M1-like macrophages differentiated using GM-CSF, while M2-like macrophages differentiated using M-CSF were poorly recognized. However, non-infected GM-CSF and M-CSF MDMs loaded with exogenous antigens elicited similar CD4+ T cell activation. IL-10 was preferentially secreted by infected M-CSF MDMs, and neutralization improved T cell activation. These results suggest that preferential infection of macrophages with an M2-like phenotype limits T cell-mediated protection against Mtb. Vaccine development should focus on T cell recognition of Mtb-infected macrophages.

Impact of Mycobacterium tuberculosis Glycolipids on the CD4+ T Cell-Macrophage Immunological Synapse.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

Workshop-based learning and networking: a scalable model for research capacity strengthening in low- and middle-income countries.

Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

Unlocking the health system barriers to maximise the uptake and utilisation of molecular diagnostics in low-income and middle-income country setting.

BACKGROUND: Early access to diagnosis is crucial for effective management of any disease including tuberculosis (TB). We investigated the barriers and opportunities to maximise uptake and utilisation of molecular diagnostics in routine healthcare settings. METHODS: Using the implementation of WHO approved TB diagnostics, Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) and Line Probe Assay (LPA) as a benchmark, we evaluated the barriers and how they could be unlocked to maximise uptake and utilisation of molecular diagnostics. RESULTS: Health officers representing 190 districts/counties participated in the survey across Kenya, Tanzania and Uganda. The survey findings were corroborated by 145 healthcare facility (HCF) audits and 11 policy-maker engagement workshops. Xpert MTB/RIF coverage was 66%, falling behind microscopy and clinical diagnosis by 33% and 1%, respectively. Stratified by HCF type, Xpert MTB/RIF implementation was 56%, 96% and 95% at district, regional and national referral hospital levels. LPA coverage was 4%, 3% below culture across the three countries. Out of 111 HCFs with Xpert MTB/RIF, 37 (33%) used it to full capacity, performing ≥8 tests per day of which 51% of these were level five (zonal consultant and national referral) HCFs. Likewise, 75% of LPA was available at level five HCFs. Underutilisation of Xpert MTB/RIF and LPA was mainly attributed to inadequate-utilities, 26% and human resource, 22%. Underfinancing was the main reason underlying failure to acquire molecular diagnostics. Second to underfinancing was lack of awareness with 33% healthcare administrators and 49% practitioners were unaware of LPA as TB diagnostic. Creation of a national health tax and decentralising its management was proposed by policy-makers as a booster of domestic financing needed to increase access to diagnostics. CONCLUSION: Our findings suggest higher uptake and utilisation of molecular diagnostics at tertiary level HCFs contrary to the WHO recommendation. Country-led solutions are crucial for unlocking barriers to increase access to diagnostics.

Qualitative assessment of the impact of socioeconomic and cultural barriers on uptake and utilisation of tuberculosis diagnostic and treatment tools in East Africa: a cross-sectional study.

OBJECTIVES: Early diagnosis and timely treatment are key elements of a successful healthcare system. We assessed the role of socioeconomic and cultural norms in accelerating or decelerating uptake and utilisation of health technologies into policy and practice. SETTING: Secondary and tertiary level healthcare facilities (HCFs) in three East African countries. Level of HCF was selected based on the WHO recommendation for implantation of tuberculosis (TB) molecular diagnostics. PARTICIPANTS: Using implementation of TB diagnostics as a model, we purposively selected participants (TB patients, carers, survivors, healthcare practitioners, community members, opinion leaders and policy-makers) based on their role as stakeholders. In-depth interviews, key informant interviews and focus group discussions were held to collect the data between 2016 and 2018. The data were transcribed, translated, coded and analysed by thematic-content analysis. RESULTS: A total of 712 individuals participated in the study. Socioeconomic and cultural factors such as poverty, stigma and inadequate knowledge about causes of disease and available remedies, cultural beliefs were associated with low access and utilisation of diagnostic and treatment tools for TB. Poverty made people hesitate to seek formal healthcare resulting in delayed diagnosis and resorting to self-medication and cheap herbal alternatives. Fear of stigma made people hide their sickness and avoid reporting for follow-up treatment visits. Inadequate knowledge and beliefs were fertile ground for aggravated stigma and believing that diseases like TB are caused by spirits and thus cured by spiritual rituals or religious prayers. Cultural norms were also the basis of gender-based imbalance in accessing care, 'I could not go to hospital without my husband's permission', TB survivor. CONCLUSION: Our findings show that socioeconomic and cultural factors are substantial 'roadblocks' to accelerating the uptake and utilisation of diagnostic and treatment tools. Resolving these barriers should be given equal attention as is to health system barriers.

Variation in the Human Leukocyte Antigen system and risk for endemic Burkitt lymphoma in northern Uganda.

Endemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma associated with Plasmodium falciparum (Pf) malaria and Epstein-Barr virus (EBV) infections. Variation in the Human Leukocyte Antigen (HLA) system is suspected to play a role, but assessments using less accurate serology-based HLA typing techniques in small studies yielded conflicting results. We studied 200 eBL cases and 400 controls aged 0-15 years enrolled in northern Uganda and typed by accurate high-resolution HLA sequencing methods. HLA results were analyzed at one- or two-field resolution. Odds ratios and 95% confidence intervals (aOR, 95% CI) for eBL risk associated with common HLA alleles versus alleles that were rare (<1%) or differed by <2% between the cases and controls as the reference category, were estimated using multiple logistic regression adjusting for age, sex, microgeography, region, malaria positivity and treatment history, and genetic variants associated with eBL. Compared to the controls, eBL cases had a lower frequency of HLA-A*02 (aOR = 0·59, 95% CI 0·38-0·91), HLA-B*41 (aOR = 0·36, 95% CI 0·13-1·00), and HLA-B*58 alleles (aOR = 0·59, 95% CI 0·36-0·97). eBL cases had a lower frequency of HLA-DPB1 homozygosity (aOR = 0·57, 95% CI 0·40-0·82) but a higher frequency of HLA-DQA1 homozygosity (aOR = 2·19, 95% CI 1·42-3·37). Our results suggest that variation in HLA may be associated with eBL risk.

Sero-diagnosis of Active Mycobacterium tuberculosis Disease among HIV Co-infected Persons using Thymidylate Kinase based Antigen and Antibody Capture Enzyme Immuno-Assays.

BACKGROUND: Clinical and laboratory diagnosis of Active Tuberculosis (ATB) and latent Mycobacterium Tuberculosis (M. tuberculosis) infections (LTBI) among people living with HIV/AIDS (PLWHA) presents formidable challenges. In the past, WHO issued an advisory against the use of existing TB sero-diagnostics. Emerging evidence, however, points to a precision of TB sero-diagnostics based on secretory rather than structural M. tuberculosis antigens. We hypothesized that secretory levels of M. tuberculosis thymidylate kinase (TMKmt) can Designate ATBI from LTBI and no TB (NTB). Here, we report in-house validation studies of levels of TMKmt antigen (Ag) and host specific TMKmt antibody (Ab) amongst HIV +ve and HIV -ve participants. METHODS AND RESULTS: Direct TMKmt Ag and host specific IgG Ab detection EIAs were conducted on broadly consented, stored serum (N=281[Ag] vs. 214 [Ab] respective) samples stratified as either HIV +ve or HIV-ve ATB relative to LTBI and No TB. On one hand, UG-peptide 1 and its PAb-based EIAs accurately diagnosed ATB relative to LTBI and NTB among HIV +ve subjects {irrespectively: (a) Ag detection ATB=OD>0.490; 95% CI: 0.7446 to 0.8715 vs. LTBI=OD<0.490; 95% CI 0.4325 to 0.4829 vs. NTB=OD<0.26; 95% CI 0.1675 to 0.2567 and (b) TMKmt specific IgG detection ATB=OD>1.00; 95% CI 1.170 to 1.528 [HIV +ve] and 2.044 to 2.978 [HIV -ve] respectively vs. LTBI=OD<1.00; 95% CI 0.2690 to 0.6396 vs. NTB=OD<; 95% CI 0.1527 to 0.8751}. HIV -ve ATB presented with Ag levels greater than NTB and less than LTBI (i.e. ATB -ve=<0.490 ODs>0.26), but displayed better ant-TMKmt IgG responses (OD>2.00; 95% CI 2.044 to 2.978) relative to HIV +ve ATB (OD<1.600; 95% CI 1.170 to 1.528); suggesting a better control of M. tuberculosis-septicemia. On the other hand, UG-peptide 2 and its PAb-based EIAs did not demonstrate ATB diagnostic potential regardless of HIV sero-status, except towards designating NTB. CONCLUSIONS: TMKmt Ab and Ag detecting EIAs based on UG-peptide 1 and its derivative PAb can accurately demarcate ATB from LTBI and NTB among HIV +ve subjects.

Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays.

BACKGROUND: Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read-thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. METHODS AND RESULTS: Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × 10-4-1 × 10-5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × 105) patient sample. CONCLUSIONS: Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.

Nurses' knowledge, practices, and barriers in care of patients with pressure ulcers in a ugandan teaching hospital.

Pressure ulcers have been identified as a major burden of hospitalization worldwide, and nurses are at the forefront of prevention. The purpose of this study was to determine the nurses' knowledge and practices regarding risk factors, prevention, and management of pressure ulcers at a teaching hospital in Uganda. The study employed a descriptive cross-sectional design. Fifty-six Ugandan registered practicing nurses were sampled. A composite self-administered questionnaire and an observation checklist were utilized. The nurses had limited knowledge about critical parameters of pressure ulcers. Prevention practices were observed to be unreliable and uncoordinated related to a significant shortage of staff and logistics for pressure ulcer prevention. Nurses had poor access to current literature on pressure ulcer prevention. Translation of nurses' knowledge into practice is possible if barriers like staff shortage, pressure relieving devices provision, and risk assessment tools are addressed at Mulago.